Promotion work for Scientific Degree of Dr.biol. (in Biotechnology)
Ainars Skangals
Cloning and expression of human atrial natriuretic factor
in bacteria Escherichia coli.
University of Latvia, 1997
Summary
The promotion work - a serie of scientific papers - was worked out 1993 -1997.
in the Biomedical Research and Study Centre, University of Latvia.
In the work a important for medicine peptide hormone - biosynthesis and
isolation of 28 amino acids long human atrial natriuretic factor (α-hANF) was
done using recombinant DNA technology. A sequence of synthetic α -hANF
gene optimized for expression in bacteria Escherichia coli was obtained with at
5'-end linker coding for Ile-Asp-Lys sequence cleavable with Lys-C
endopeptidase. Gene was cloned into the vector pUC8 after that insert selected
by DNA sequencing was recloned into the vector pFRCP8133, where carrier
bacteriophage fr coat protein (fr CP) gene was placed under the control of
tryptophan promoter. In this manner the expression vector pFAN15 for fusion
protein fr CP- α -hANF stable against degradation with cell proteases was
obtained.
Cultivation of recombinant E. coli bacteria in shake flasks as well as bath highcell-density culture in bioreactor was optimized resulting in fusion protein yield
more than 15% of total cell protein. Fusion protein from solubilized E. coli cells
was purified to homogenity by ion exchange chromatography on DEAE-, CMcellulose, QAE Sephadex A25 columns and selective precipitation. Hybrid fr CPα -hANF cleavage with Lys-C endopeptidase was performed with efficiency up to
40%, products of hydrolysis were fractioned by high performance liquid
chromatography (HPLC) on reversed phase column and purified α -hANF
peptide was physico-chemically and biologically characterized.
Starting sequence of hybrid gene was modified also by site-directed
mutagenesis using pFAN15 plasmid as template for change cleavage site for
lysine to tryptophan residue. This modification allows to isolate native α -hANF
by more technologic chemical cleavage with 3-bromo-3-methyl[(2nitrophenyl)thiol]-3H-indole (BNPS-skatole). The expression of modified himeric
protein in shake flask culture was studied and growth dynamics of selected
clone RR1/pTANF12 and initial RR1/pFAN15 were compared. The modified
fusion protein was isolated and purified by previosly described methods. It was
found that mutation introduced practically don't change fusion protein features.
Purified fr CP-Trp-α -hANF protein was cleaved with BNPS-skatole and products
were characterized, although efficiency of cleavage obtained until now - 2-5% is
not sufficient to isolate preparative amounts of peptide.
Results of the current work were published in four articles in international
scientific journals and a book, a patent registered in the republic of South Korea
and were reported at four international conferences.
List of original papers
1. Berzins, V., Jansone, I., Skangals, A., Kalnins, P., Liepa, S. and Baumanis, V.
(1993). High level expression ofa-human atrial natriuretic factor as a fusion
polypeptide with phage fr coat protein in Escherichia coli. J. Biotechnology,
30, 231-143.
2. Berzins, V., Baumanis, V., Skangals, A., Mandrika, I. and Jansone, I. (1994).
Isolation of Human α -Atrial Natriuretic Factor (α -hANF) from Recombinant
Fusion Protein" In: Separations/or Biotechnology 3, D.L. Pyle (ed.), Thomas
Graham House, Science Park, Cambridge, UK, 73-78.
3. Berzins V., Jansone I., Skangals A., Liepa S., Baumanis V., Kozlovska T.,
Pusko P., Gren E. (1994). A method for the microbial production of human aatrial natriuretic factor (a-hANF) and vectors for use therein. South Korea
Patent Application Nr 15002686 from 16.03.94
4. Mandrika, I., Baumanis, V., Jansone, I., Skangals, A. and Berzins, V. (1997).
Development of immunoenzymatic reactions of peptide using recombinant
hybrid proteins. Biochemistry (Moscow), 62, 217-220.
5. Baumanis, V., Jansone, I., Skangals, A., Mandrika, I. and Berzins, V. (1997).
Recombinant atrial natriuretic peptide (rANP) from hybrid fused protein phage fr coat /ANP (CP/ANP). Peptides, 18, (in press).